Preparation of l-isoleucine by fermentation

ABSTRACT

L-isoleucine is prepared by culturing a hydrocarbon assimilable microorganism in a culture medium containing hydrocarbon as the main carbon source and Alpha -aminobutyric acid, hydroxybutyric acid, threonine or mixtures thereof or non-toxic salts thereof. Microorganisms of the genera Corynebacterium, Brevibacterium, Arthrobacter, Micrococcus, Pseudomonas, Nocardia, Streptomyces, etc., are particularly suitable for use in the process.

United States Patent Nakayama et al. 1 June 20, 1972 54] PREPARATION OF L-ISOLEUCINE BY 3,219,543 ll/l965 Douros et al 1 95/3 x FERMENTATION 3,222,258 12/1965 lizuka et al ..l95/3 X [72] inventors: Kiyoshi Nakayama, Sagamihara-shi; OTHER PUBLICATIONS gl z? Hagmo Hachlojl'sh" both of Ajinomoto Co., Prep. of lsoleucine by Fermentation,"

p Chem. Abs., Vol. 63, 1965, No. 3590. [73] As i ee; Kyo a Hakko Kogyo K b hiki K i h Otsuka et al.,J. Gen. Appl. Microbiol, Vol. 12, p. ll l, 1966. Tokyo-to,j Wada, H., Isoleucine, Chem. Abs., Vol. 64, No. 4229, 1966. [221 March 5, 1969 Yamada et al., Agr. Biol. Chem. Vol. 27, pp. 390- 39s, 1963.

[211 App]. No.: 804,696 I Primary Exammer-A. Louis Monacell Assistant Examiner-Gary M. Nath [30] Foreign Application Priority Data Attomey-Bacon & Thomas March 13, 1968 Japan ..43/l5903 [57] ABSTRACT 521 US. Cl. ..195/28 R h-isoleucine is Prepared y culturing a hydrocarbon assimila- [51] Int. Cl. ..Cl2d 13/06 ble micrmrganism in a Culture medium containing y 58 Field ofSeai-ch ..195/3 H,28,30,41-70, as the main carbon Source and ol-aminobuwfl'c acid. 195/29 hydroxybutyric acid, threonine or mixtures thereof or nontoxic salts thereof. Microorganisms of the genera Corynebac- [56] References Cited terium, Brevibacterium, Arthrobacter, Micrococcus, Pseu domonas, Nocardia, Streptomyces, etc., are particularly suita- UNITED STATES PATENTS ble for use in the Process.

3,058,888 10/ 1962 Chibata et al 195/30 13 Claims, No Drawings PREPARATION OF L-ISOLEUCINE BY FERMENTATION BACKGROUND OF THE INVENTION L-isoleucine is an essential amino acid and is useful as a nutrient for humans and animals. It is possible, for example, to improve the nutritional value of proteins by addition of L- isoleucine.

Prior investigators have reported that L-isoleucine can be obtained by fermentation by employing a culture medium mainly containing saccharides. Additionally, the substitution of less expensive hydrocarbons for saccharides has been reported [U.S. Pat. No. 3,222,258] but only small amounts of L- isoleucine were reported as being accumulated in the culture medium [about 2.2 mg/l according to the examples of US. Pat. No. 3,222,258]. The reported processes, therefore, are not well suited for the commercial production of L-isoleucine.

DETAILED DESCRIPTION OF THE INVENTION We have discovered that L-isoleucine can be obtained in large yield by culturing a hydrocarbon assimilable microorganism in a culture medium containing hydrocarbon as the main carbon source and, in addition, a-aminobutyric acid, hydroxybutyric acid, threonine or mixtures thereof or nontoxic salts thereof.

Any hydrocarbon assimilable microorganism capable of producing L-isoleucine may be used for the purpose of the present invention. Microorganisms capable of producing L- isoleucine according to the process of this invention are found in various genera but particularly suitable microorganisms are members of the genera Corynebacterium, Brevibacterium, Arthrobacter, Micrococcus, Pseudomonas, Candida, Nocardia, Streptomyces, etc.

The action and function of a-aminobutyric acid, a-hydroxybutyric acid, acid, threonine or mixtures thereof is not sufi'iciently known to determine whether they act as substrates for the preparation of L-isoleucine. Even if they do act as a substrates, the present invention provides the use of cheaper hydrocarbon sources than those conventionally employed for the same purpose and, accordingly, provides a process more easily adopted for commercial use.

The effect of the addition of a-aminobutyric acid on a hydrocarbon-containing culture medium with respect to L- isoleucine fermentation was determined by using an Lisoleucine-producing strain belonging to the genus Corynebacterium hydrocarboclastus. a-Aminobutyric acid in an amount shown in Table l was added to 10 ml each of a culture medium containing an n-alkane mixture (C -C (2 g/dl), Nl-LCI (0.34 g/dl), KI-I PO (0.16 g/dl), MgSO,,- 7h (0.05 g/dl), NaCl (0.2 g/dl), thiamine hydrochloride (50 mg/l) and having a pH of 7.2.

After the a-aminobutyric acid was placed in the culture media, the media were each placed in 250 ml Erlenmeyer flasks. The media were sterilized at 115 C. for 10 minutes. Each medium was then innoculated with the aforementioned strain and cultured at 30 C. for 96 hours with shaking (230 rpm.) by using a conventional shaker to produce the quantity of L-isoleucine shown in Table 1. From this table, it is apparent that the accumulation of L-isoleucine is largely increased by addition of a-aminobutyric acid.

The amount of a-aminobutyric acid added to any given culture medium will vary depending upon the type of microorganism employed, the composition of the medium and the concentration thereof, etc., but it is preferred to use 0.5-50g/l.

Similar effects to those disclosed in Table 1 were also found when a-hydroxybutyric acid, threonine or mixtures thereof were added to the culture medium.

Any synthetic or natural medium containing a-aminobw tyric acid, a-hydroxybutyric acid, threonine or mixtures thereof or non-toxic salts thereof and suitable amounts of assimilable hydrocarbon as the main carbon source, nitrogen source, inorganic substances and other nutrients necessary for the growth of the microorganism employed may be used for the purpose of this invention.

As the hydrocarbon source in the culture medium, various assimilable hydrocarbon sources such as kerosene, gasoline, heavy oil, butane, naptha, ligroin, natural gas, propane, ethane, methane, etc., may be used.

As the nitrogen source, both organic and inorganic nitrogen compounds, such as urea, nitrates, ammonia and ammonium salts, e.g., ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium carbonate, ammonium acetate, etc., may be used. Various other nitrogen-containing substances may also be used as well as peptone, meat extract, yeast extract, com steep liquor, casein hydrolysate, fish meal, soybean meal, etc. These may be used solely or in combination with other substances.

As the inorganic substances which may be included in the culture medium, there may be mentioned K I-IPO KH PO magnesium sulfate, zinc sulfate, manganese sulfate, calcium carbonate, etc.

In addition, vitamins, amino acids and bases, etc., may be added to the culture medium according to characteristics of the microorganisms employed.

The fermentation should be carried out under aerobic conditions such as by means of aeration, shaking, etc. The culturing temperature is preferably 20 to 40 C. The pH of the medium may vary but it is advantageous to adjust the pH to about neutral with acid or alkali. 1 1

The following examples serve to illustrate the invention, but they are not to be considered as limiting the same:

EXAMPLE 1 Fermentation was carried out using a culture medium containing an n-alkane mixture (C -C (l g/dl), (NI-[0 80 (2g/dl), Kl-l PO, (0.2 g/dl), NaHlO (0.2 g/dl). MgSO,,'7I-I,0 (0.1 g/dl), FeSO; 7H 0 (0.001 g/dl), MnSO -4H O (0.001 g/dl). ZnSO '2H O (0.001 g/dl), thiamine (1 mg/l), yeast extract (0.3 g/dl) CaCO; (2 gldl) and DL-a-aminobutyric acid (IOmg/ml) and having a pH of 7.4. Ten ml portions portions of the media were separately placed in large test tubes and each innoculated with one of the microorganisms shown in Table 2. Fermentation was carried out with shaking at 30 C. for 96 hours to obtain the quantity of L-isoleucine shown in Table 2. Under similar culture conditions but in the absence of DL-a-aminobutyric acid, no substantial quantity of L-isoleucine was accumulated except with Corynebacterium hydrocarboclastus (ATCC 15592) (50 mg/l).

The accumulated L-isoleucine was recovered by using ion exchange resin in known manner.

TABLE I TABLE 2 a-Aminobutyric acid added Amount of L-isoleucine Amount f i 1 i (mg/ml) Accumulated g/ Microorganism Produced g/l) 0 10 Arlhmbacter hydmcarbogluramicus 1 200 (ATCC 15583) 1.00 10 4600 Micrococcus parafinoliticus 30 4700 (ATCC 15582) 1.55

Corynebacten'um hydrocarboclasms (ATCC 15592) 1.50

Pseudomona: aeruginora (ATCC 7700) 0.10 Pseudomdnas Vendoreli (lFO 3899) 0.05 Candida tropicalis (1AM 4924) 40 Candida lipolytica (IFO 0746) 0,10 Nocardia glaberula (ATCC 21022) 0.20 S treptom yces antibiolicus (ATCC 10382) 0.30

EXAMPLE 2 Microorganisms shown in Table 3 were cultured in a manner similar to that d described in Example 1 with the exception that DL-a-hydroxybutyric acid (5 mg/ml) was used in place of a-aminobutyric acid. The results obtained are shown in Table'3. 7

TABLE, 3

Amount of L-isoleucine Microorganism Produced g/l) Microcvccus paraffinoliticus (ATCC 15582) 0.50 Micrococcus para ffineus (ATCC 15589) 0.25 C orynebaclerium hydrocarboclaslus (ATCC 15592) 0.75 Nocardia globerula (ATCC 21022) Cultivation similar to that described above was performed without adding a-hydroxyburyric acid to the media. No substantial quantity of L-isoleucine was accumulated except with C orynebacterium hydracarboclastus (ATCC 15592) (50 mg/l).

EXAMPLE 3 TABLE 4 Amount of L-isoleucine Microorganism Produced (g/l) A rthrobacter It ydrocarboglulamicus (ATCC 15583) 0.15 Micrococcus paraffinolilicus (ATCC 15582) 0.25 C orynebaclerium hydrocarboclastus (ATCC 15592) 0.25 Preudomonar Vendoreli (lFO 3899) 0.10

EXAMPLE 4 Microorganisms shown in Table 5 were cultured in a manner similar to that described in Example 1 with the exception that L-thrconine (10 mg/ml) was substituted for aaminobutyric acid to give the results shown in Table 5. Cultivation similar to that described above was performed without addition of L-threonine to the media. No substantial quantity of L-isoleucine was accumulated except with Corynebacterium hydr0carbT0Xll3zsLtgs5(ATCC 15592) (50 mg/l).

Amount of L-isoleucine .of said culture medium A culture of each organism identified by ATCC (American 7 Type Culture Collection), 1AM (institute of Applied Microbiology, Tokyo University, Tokyo, Japan) number or [F0 (Institute for Fermentation, Osaka, Japan) has been deposited without restriction as to its being made available to the public at the identified depository.

We claim: v

1. A process of preparing L-isoleucine by fermentation, comprising aerobically culturing a hydrocarbon-assimilable microorganism selected from the group consisting of Arlhrobacter hydrocarboglutamicus ATCC 15583, Microcaccus paraffinoliticus ATCC 15582, Micrococcus paraffirteus ATCC 15589, Corynebacterium hydrocarboclaslus ATCC 15592, Pseudomonas aeruginosa ATCC 7700, Pseudomonas vendoreli lFO 3899, Nocardia globerula ATCC 21022, Streptomyces antibiolicus ATCC 10382 and Brevibacterium ketoglutamicum ATCC 15587 in a culture medium containing hydrocarbon as the main carbon source and a-aminobutyric acid, a-hydroxybutyric acid, threonine or mixtures thereof or non-toxic salts thereof and recovering theaccumulated L- isoleucine from said culture medium.

2. A process as claimed in claim 1 in which the temperature is maintained within the range of 20-40 C.

3. A process as claimed in claim 1 in which the pH of said culture medium is maintained at approximately 7.

4. A process as claimed in claim 1 in which said culture medium contains a nitrogen source, inorganic material and nutrients.

5. A process as claimed in claim 1 in which the microorganism is Arthrobacter hydrocarboglutamicus ATCC 15583. 7

6. A process as claimed in claim 1 in which the microorganism is Micrococcus paraffinoliticus ATCC 15582.

7. A process as claimed in claim 1 in which the microorganism is Micrococcus paraffineus ATCC 15589.

8. A process as claimed in claim 1 in which the microorganism is Corynebacterium hydrocarboclastus ATCC 15592.

9. A process as claimed in claim 1 in which the microorganism is Pseudomonas aeruginosa ATCC 7700.

10. A process as claimed in claim 1 in which the microorganism is Pseudomonas vendoreli IFO 3899.

11. A process as claimed in claim 1 in which the microorganism is Nocardia globerula ATCC 21022.

12. A process as claimed in claim 1 in which the microorganism is Streptomyces antibioticus ATCC 1038 2.

13. A process as claimed in claim 1 in which the microorganism is Brevibacrerium keloglulamicum ATCC 15587.

a t at a 

2. A process as claimed in claim 1 in which the temperature of said culture medium is maintained within the range of 20*-40* C.
 3. A process as claimed in claim 1 in which the pH of said culture medium is maintained at approximately
 7. 4. A process as claimed in claim 1 in which said culture medium contains a nitrogen source, inorganic material and nutrients.
 5. A process as claimed in claim 1 in which the microorganism is Arthrobacter hydrocarboglutamicus ATCC
 15583. 6. A process as claimed in claim 1 in which the microorganism is Micrococcus paraffinoliticus ATCC
 15582. 7. A process as claimed in claim 1 in which the microorganism is Micrococcus paraffineus ATCC
 15589. 8. A process as claimed in claim 1 in which the microorganism is Corynebacterium hydrocarboclastus ATCC
 15592. 9. A process as claimed in claim 1 in which the microorganism is Pseudomonas aeruginosa ATCC
 7700. 10. A process as claimed in claim 1 in which the microorganism is Pseudomonas vendoreli IFO
 3899. 11. A process as claimed in claim 1 in which the microorganism is Nocardia globerula ATCC
 21022. 12. A process as claimed in claim 1 in which the microorganism is Streptomyces antibioticus ATCC
 10382. 13. A process as claimed in claim 1 in which the microorganism is Brevibacterium ketoglutamicum ATCC
 15587. 